supplemental materials - Sulzer lab, Columbia University

Supporting information
Development of pH-Responsive Fluorescent False Neurotransmitters
Minhee Lee,1 Niko G. Gubernator,2 David Sulzer,3,* and Dalibor Sames1,*
1
Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027,
USA. 2eMolecules, San Diego, CA 92014, 3Department of Neurology, Psychiatry and
Pharmacology, Columbia University, College of Physicians and Surgeons, New York, New York
10032, USA.
Part I: Structure and Synthesis of Coumarin Probes
Part II: HPLC Analysis of Coumarin Probes
Part III: Photophysical Characterization and Measurement of log D values
Part IV: Protocols for Fluorescence Microscopy Assay in HEK and VMAT2-HEK
cells and Assay Results Summary
Part V: Protocols for Fluorescence Microscopy in PC-12 cells
Part VI: Protocols for Two-photon Fluorescence Microscopy and pH Measurement
of Secretory Vesicles in PC-12 Cells
Part VII: Preparation and Uptake Test of the Hydrochloride Salt of Mini202
Part VIII: 1H and 13C NMR of Coumarin Probes
S1 Part I: Structure and Synthesis of Coumarin Probes
General
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich or Strem and
used without further purification. When necessary, solvents were dried by passing them
through a column of alumina under argon. Flash chromatography was performed on
SILICYCLE silica gel (230–400 mesh). Nuclear Magnetic Resonance spectra were
recorded at 300 K (unless otherwise noted) on Bruker 300 or 400 Fourier transform NMR
spectrometers. Proton chemical shifts are expressed in parts per million (ppm, δ scale)
and are referenced to residual protium in the NMR solvent (CDCl3, δ 7.26; CD3OD, δ
3.30; DMSO, δ 2.49). Data for 1H NMR are reported as follows: chemical shift,
integration, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, bs = broad
singlet), and coupling constant in Hertz (Hz). Carbon chemical shifts are expressed in
parts per million (ppm, δ scale) and are referenced to the carbon resonance of the NMR
solvent (CDCl3, δ 77.0; CD3OD, δ 49.0; DMSO, δ 39.5). Mass spectra were recorded on
a JEOL LCmate (ionization mode: APCI+) or on a JMSHX110 HF mass spectrometer
(ionization mode: FAB+). Preparative HPLC was performed with a Waters 600 Controller
on a Vydac C18 Protein & Peptide column (#218TP1022); fractions were detected with a
Waters 2487 Dual λ Absorbance Detector and collected with a Waters 2767 Sample
Manager. Data was analyzed using OpenLynx software. Isocratic elution or linear
gradients of solvents A and B were used (A = HPLC grade acetonitrile or methanol
containing 10% (v/v) de-ionized water (Millipore Simplicity 185, 18.2 MΩ); B = deionized water containing 0.1% (v/v) trifluoroacetic acid (ReagentPlus grade, 99%).
Analytical HPLC was performed on a Vydac C18 Protein & Peptide column (#218TP54).
S2 Figure S1. Structure of coumarin probes as trifluoroacetic acid salts
S3 Scheme S1. Synthesis of Mini101, 102, 103, 104, 105, and 106. (a) MSA (Methanesulfonic acid), RT, 2 h,
63%, 28%, and 5% for Mini101, 104, and 105, respectively; (b) MSA, RT, 3 h, 37%; (c) MSA, RT, 3 h,
51% and 3% for Mini103 and Mini106, respectively.
Compound series of Mini101–106 were synthesized via a von Pechmann type
condensation
methods)
1,2
of resorcinols with β-ketoester 1 or 2 (prepared by following the published
in methanesulfonic acid3,4 for 2–3 h at RT followed by purification by RP-
HPLC to obtain the probes as TFA salts. Synthesis of Mini102 is described below as a
representative example.
To a mixture of 4-chlororesorcinol (0.17 g, 1.2 mmol) and 1 (0.20
g, 0.8 mmol) was added methanesulfonic acid (1.3 mL, 20 mmol)
at 0 °C. The clear brown solution gradually became dark orange
within 3 h at which point the reaction mixture was diluted with
cold ethyl ether (−20 °C, 10 mL), and centrifuged (3000 rpm) at 4 °C for 20 min. After
removing the ether solvent by aspiration, the residual orange solid was dried under high
vacuum, dissolved in H2O (3 mL), and purified by RP-HPLC using an appropriate linear
gradient of acetonitrile containing 10% de-ionized water (A) and 0.1% (v/v) TFA/H2O
1
Jones, R. C. F.; Bhalay, G.; Carter, P. A.; Duller, K. A. M.; Dunn, S. H. J. Chem. Soc., Perkin Trans. 1,
1999, 765-776.
2
Moreau, R. J.; Sorensen, E. J. Tetrahedron 2007, 63, 6446-6453.
3
Sun, W. C.; Gee, K. R.; Haugland, R. P. Bioorg. Med. Chem. Lett. 1998, 8, 3107-3110.
4
Brun, M.-P.; Bischoff, L.; Garbay, C. Angew. Chem., Int. Ed. 2004, 43, 3432-3436.
S4 (B) (3–50% A over 20 min followed by a steep gradient to 100% A and equilibrium back
to 3% A). The fractions containing the product (retention time ~12.7 min) were collected,
concentrated, and lyophilized to give Mini102 as a white solid (37%). Alternatively, for
larger scale synthesis of Mini102, the residue resulting from precipitation by ethyl ether
was recrystalized from 0.1 M aq. HCl (twice) to give Mini102HCl as a pale pink solid.
1
H NMR (DMSO-d6, 300 MHz): δ 11.55 (1H, bs), 7.86 (1H, s), 7.86 (3H, bs), 6.95 (1H,
s), 6.25 (1H, s), 3.11 (2H, t, J = 6.2 Hz ), 3.03 (2H, t, J = 6.0 Hz). 13C NMR (DMSO-d6,
75 MHz): δ 160.5, 157.4, 154.3, 151.9, 126.4, 118.0, 113.4, 112.5, 104.5, 38.4, 29.7.
LRMS (APCI+): Calc’d for C11H10ClNO3 239.0 m/z, measured 240.2 (MH+).
Mini101
1
H NMR (DMSO-d6, 300 MHz): δ 10.65 (1H, s), 7.84 (3H, bs), 7.65 (1H, d, J = 8.8 Hz),
6.83 (1H, dd, J = 8.7, 2.4 Hz), 6.75 (1H, d, J = 2.3 Hz), 6.18 (1H, s), 3.19 – 3.09 (2H, m),
3.02 (2H, t, J = 6.8 Hz).
13
C NMR (DMSO-d6, 75 MHz): δ 162.2, 161.0, 156.1, 152.7,
127.0, 113.9, 112.1, 111.7, 103.4, 38.3, 29.8. LRMS (APCI+): Calc’d for C11H11NO3
205.1 m/z, measured 206.1 (MH+).
Mini103
1
H NMR (DMSO-d6, 300 MHz): δ 11.26 (1H, bs), 7.93 (3H, s), 7.68 (1H, d, J = 11.8 Hz),
6.95 (1H, d, J = 7.5 Hz), 6.26 (1H, s), 3.12 (2H, t, J = 6.6 Hz), 3.01 (2H, t, J = 6.6 Hz).
13
C NMR (DMSO-d6, 75 MHz): δ 159.9, 151.4, 150.8, 149.2 (d, J = 14.4 Hz), 148.2 (d, J
= 238.1 Hz), 112.6, 111.2 (d, J = 21.4 Hz), 110.2 (d, J = 7.3 Hz), 104.9, 37.5, 29.0.
LRMS (APCI+): Calc’d for C11H10FNO3 223.1 m/z, measured 224.3 (MH+).
Mini104
1
H NMR (DMSO-d6, 300 MHz): δ 10.73 (1H, s), 7.88 (3H, bs), 7.40 (1H, s), 6.79 (1H, s),
6.15 (1H, s), 3.22 – 3.12 (2H, m), 3.07 (2H, t, J = 6.9 Hz), 1.39 (9H, s).
13
C NMR
(DMSO-d6, 75 MHz): δ 160.2, 160.0, 153.2, 152.0, 133.3, 121.9, 110.7, 109.9, 103.1,
37.3, 34.4, 29.2, 28.6. LRMS (APCI+): Calc’d for C15H19NO3 261.1 m/z, measured 262.1
(MH+).
S5 Mini105
1
H NMR (DMSO-d6, 300 MHz): δ 10.67 (1H, s), 7.83 (3H, bs), 7.40 (1H, s), 6.74 (1H, s),
6.15 (1H, s), 3.22 – 3.03 (4H, m), 1.96 (2H, s), 1.43 (6H, s), 0.72 (9H, s).
13
C NMR
(DMSO-d6, 75 MHz): δ 160.5, 160.1, 153.2, 152.0, 132.5, 122.8, 110.6, 110.0, 102.9,
51.2, 38.5, 37.3, 32.0, 31.2, 30.8, 28.6. LRMS (APCI+): Calc’d for C19H27NO3 317.2 m/z,
measured 318.0 (MH+).
Mini106
1
H NMR (CD3OD, 300 MHz): δ 7.49 (2H, d, J = 11.2 Hz), 7.37 – 7.34 (2H, m), 7.23 –
7.12 (3H, m), 6.26 (1H, s), 4.20 (2H, s), 3.27 (2H, t, J = 7.8 Hz), 3.10 (2H, t, J = 7.5 Hz).
13
C NMR (CD3OD, 75 MHz): δ 162.7, 153.3, 150.6, 150.2 (d, J = 256.7 Hz), 148.7 (d, J
= 3.6 Hz), 141.0, 129.7, 129.2, 127.1, 120.2, 113.4, 111.7 (d, J = 7.7 Hz), 109.2 (d, J =
22.4 Hz), 39.1, 30.5, 29.6. LRMS (APCI+): Calc’d for C18H16FNO3 313.1 m/z, measured
314.3 (MH+).
Scheme S2. Synthesis of Mini107, 108, and 109. (d) formaldehyde (~37 wt. % in H2O), NaBH(OAc)3,
CH2Cl2, RT, 6 h, 40%; (e) formaldehyde (~37 wt. % in H2O), NaBH(OAc)3, CH2Cl2, RT, 12 h, 76%; (f)
formaldehyde (~37 wt. % in H2O), NaBH(OAc)3, CH2Cl2, RT, 15 h, 51%.
Compound series of Mini107–109 were synthesized via Eschweiler-Clarke dimethylation
S6 of Mini101–103 in dichloromethane with formaldehyde and NaBH(OAc)3. The reaction
mixture was stirred for the indicated time (Scheme S2) at room temperature. Purification
by HPLC provided the probes as TFA salts. Synthesis of Mini107 is described below as a
representative example.
To Mini101 (40 mg, 0.13 mmol) in CH2Cl2 (5 mL, 0.026 M) were
added formaldehyde (~37 wt. % in H2O, 0.20 g, 2.5 mmol) and
NaBH(OAc)3 (1.3 g, 6.1 mmol). The reaction solution was stirred for
6 h at RT, and the crude mixture was extracted into H2O (2 x 2 mL)
and purified by RP-HPLC using an appropriate linear gradient of acetonitrile containing
10% de-ionized water (A) and 0.1% (v/v) TFA/H2O (B) (3–25% A over 30 min followed
by a steep gradient to 100% A and equilibrium back to3% A). The fractions containing
the product (retention time ~18.1 min) were collected, concentrated, and lyophilized to
give Mini107 as a white solid (40%). 1H NMR (DMSO-d6, 300 MHz): δ 10.76 (1H, bs),
9.85 (1H, bs), 7.71 (1H, d, J = 8.8 Hz), 6.84 (1H, dd, J = 8.7, 2.4 Hz), 6.75 (1H, d, J =
2.3 Hz), 6.21 (1H, s), 3.40 (2H, t, J = 7.5 Hz), 3.15 (2H, t, J = 7.5 Hz), 2.87 (6H, s). 13C
NMR (DMSO-d6, 100 MHz): δ 161.5, 160.1, 155.2, 151.9, 126.4, 113.1, 110.7, 110.6,
102.6, 54.6, 42.4, 25.8. LRMS (APCI+): Calc’d for C13H15NO3 233.1 m/z, measured 234.2
(MH+)
Mini108
1
H NMR (DMSO-d6, 400 MHz): δ 11.54 (1H, bs), 9.58 (1H, bs), 7.92 (1H, s), 6.94 (1H,
s), 6.28 (1H, s), 3.47 – 3.33 (2H, m), 3.16 (2H, t, J = 8.0 Hz), 2.88 (6H, s).
13
C NMR
(DMSO-d6, 100 MHz): δ 159.6, 156.6, 153.3, 151.1, 125.8, 117.3, 111.7, 111.6, 103.6,
54.4, 42.4, 25.6. LRMS (APCI+): Calc’d for C13H14ClNO3 267.1 m/z, measured 268.2
(MH+)
Mini109
1
H NMR (DMSO-d6, 400 MHz): δ 11.23 (1H, bs), 9.51 (1H, bs), 7.75 (1H, d, J = 11.8
Hz), 6.96 (1H, d, J = 7.5 Hz), 6.30 (1H, s), 3.46 – 3.34 (2H, m), 3.13 (2H, t, J = 8.0 Hz),
2.88 (6H, s). 13C NMR (CD3OD, 75 MHz): δ162.7, 152.6, 152.5, 151.3 (d, J = 15.1 Hz),
S7 150.4 (d, J = 239.5 Hz) 113.0, 111.9 (d, J = 21.7 Hz), 111.8, 106.3 (d, J = 1.8 Hz), 56.6,
43.7, 27.5. LRMS (APCI+): Calc’d for C13H14FNO3 251.1 m/z, measured 252.2 (MH+).
Scheme S3. Synthesis of Mini201 and Mini202. (g) BnBr, NaI, NaHCO3, MeCN, 80 °C, 18 h, 49%; (h)
Boc-GABA-OH, DIC, DMAP, CH2Cl2, RT, 9 h, 90%; (i) 2,8,9-triisopropyl-2,5,8,9-tetraaza-1phosphabicyclo[3.3.3]undecane, 3Å molecular sieves, benzene, 50 °C, 2.5 h, 41%; (j) trifluoroacetic acid,
triisopropylsilane, H2O, RT, 2 h; (k) Pd/C, EtOH/MeOH, RT, 2 h, 73% for two steps; (l) SO2Cl2, Et2O, 0 °C
→ RT, 30 min, 47%; (m) BnBr, NaI, NaHCO3, MeCN, 80 °C, 16 h, 58%; (n) Boc-GABA-OH, DIC,
DMAP, CH2Cl2, RT, 2.5 h, 81%; (o) 2,8,9-triisopropyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane,
3Å molecular sieves, benzene, 50 °C, 17 h, 44%; (p) trifluoroacetic acid, triisopropylsilane, H2O, 85 °C, 16
h, 66%.
To 2,4-dihydroxybenzaldehyde (2.0 g, 14 mmol), NaI (1.0 g, 7.2 mmol),
and NaHCO3 (1.5g, 17 mmol) in acetonitrile (30 mL, 0.50 M) was added
benzyl bromide (1.7 mL, 14 mmol). The reaction mixture was stirred at
80 °C for 18 h under argon, cooled to RT, diluted with EtOAc (100 mL), washed with
H2O and brine, dried over MgSO4, and filtered. The filtrate was concentrated and
purified by flash chromatography (silica, EtOAc:hexanes = 1:8) to provided compound 3
as a white solid (49%). 1H NMR (CDCl3, 300 MHz): δ 11.48 (1H, s), 9.72 (1H, s), 7.51 –
7.32 (6H, m), 6.62 (1H, dd, J = 8.7, 2.3 Hz), 6.54 (1H, d, J = 2.3 Hz), 5.11 (2H, s). 13C
S8 NMR (CDCl3, 100 MHz): δ 194.5, 166.0, 164.6, 135.8, 135.4, 128.9, 128.5, 127.6, 115.5,
109.0, 101.8, 70.5. LRMS (APCI+): Calc’d for C14H12O3 228.1
m
/z, measured 229.2
(MH+).
To Boc-GABA-OH (0.85 g, 4.2 mmol) in dichloromethane
(35 mL) were added N,N′-diisopropylcarbodiimide (DIC,
0.70 mL, 4.6 mmol), 4-(dimethylamino)pyridine (DMAP,
0.11 g, 0.90 mmol), and compound 3 (0.80 g, 3.5 mmol) successively. The reaction
mixture was stirred at RT for 9 h, after which time the reaction mixture was washed with
H2O and brine, dried over MgSO4, filtered, and concentrated. The crude product was
purified by flash chromatography (silica, EtOAc:hexanes = 1:5 → 1:3) to give compound
4 as a white solid (90%). 1H NMR (CDCl3, 400 MHz): δ 9.91 (1H, s), 7.79 (1H, d, J =
8.7 Hz), 7.54 – 7.30 (5H, m), 6.96 (1H, dd, J = 8.6, 2.4 Hz), 6.79 (1H, d, J = 2.3 Hz),
5.13 (2H, s), 4.79 (1H, s), 3.36 – 3.15 (2H, m), 2.71 (2H, t, J = 7.2 Hz), 2.02 – 1.89 (2H,
m), 1.45 (9H, s). 13C NMR (CDCl3, 100 MHz): δ 187.8, 171.5, 164.4, 156.2, 153.1, 135.6,
133.8, 122.0, 113.0, 109.9, 79.4, 70.7, 39.7, 31.2, 28.5, 25.1.
To a solution of compound 4 (0.90 g, 2.2 mmol) in dry benzene
(2.0 mL) was added 3Å molecular sieves (powder, 2.0 g). To
this
solution,
2,8,9-triisopropyl-2,5,8,9-tetraaza-1-phospha-
bicyclo[3.3.3]undecane (0.29 g, 0.96 mmol) in dry benzene (2.0 mL) was added via
syringe at 40 °C. After being stirred at 50 °C under argon for 2.5 h, the reaction mixture
was cooled to RT and loaded directly onto a silica gel column. Purification by flash
column chromatography (silica, EtOAc:hexanes = 1:3 → 1:2) yielded compound 5 as a
white solid (41%). 1H NMR (CDCl3, 400 MHz): δ 7.45 – 7.27 (7H, m), 6.87 (1H, dd, J =
8.6, 2.4 Hz), 6.81 (1H, d, J = 2.4 Hz), 5.06 (3H, s), 3.41 – 3.37 (2H, m), 2.70 (2H, t, J =
5.2 Hz), 1.39 (9H, s). 13C NMR (CDCl3, 100 MHz): δ 162.5, 161.5, 156.4, 155.3, 140.7,
136.3, 129.1, 128.9, 128.7, 127.9, 123.5, 113.6, 113.5, 101.9, 79.5, 70.8, 39.6, 31.9, 28.8.
LRMS (APCI+): Calc’d for C23H25NO5 395.2 m/z, measured 396.3 (MH+).
S9 Compound 5 (0.36 g, 0.88 mmol) was treated with
trifluoroacetic acid (5 mL), triisopropylsilane (0.15 mL),
and H2O (0.15 mL) at RT for 2 h, after which time the
solvent was removed under reduced pressure and Et2O was added to precipitate the Bocdeproteced compound as a white solid. After filtration, the residue was dried under high
vacuum, dissolved in EtOH/MeOH (10 mL/10mL), and treated with Pd/C (25 mg). After
being stirred vigorously under H2 (50 psi) at RT for 2 h using Parr hydrogenation
apparatus, the reaction solution was filtered through celite, concentrated, and purified by
PR-HPLC using an appropriate linear gradient of acetonitrile containing 10% de-ionized
water (A) and 0.1% (v/v) TFA/H2O (B) (3–25% A over 30 min followed by a steep
gradient to 100% A and equilibrium back to 3% A). The fractions containing the product
(retention time ~16.9 min) were collected, concentrated, and lyophilized to give Mini201
as a white solid (73% for two steps). 1H NMR (DMSO-d6, 400 MHz): δ 7.89 (3H, s),
7.79 (1H, s), 7.47 (1H, d, J = 8.5 Hz), 6.80 (1H, dd, J = 8.5, 2.3 Hz), 6.73 (1H, d, J = 2.2
Hz), 3.10 – 3.05 (2H, m), 2.72 (2H, t, J = 7.0 Hz).
13
C NMR (DMSO-d6, 75 MHz): δ
161.3, 160.9, 154.8, 141.9, 129.2, 119.2, 113.2, 111.6, 101.9, 37.3, 28.6. LRMS (APCI+):
Calc’d for C11H11NO3 205.6 m/z, measured 206.1 (MH+).
To a solution of 2,4-dihydroxybenzaldehyde (3.0 g, 22 mmol) in Et2O
(100 mL, 0.22 M) was added dropwise sulfurylchloride (2.1 mL, 26
mmol) at 0 °C under argon. After being stirred at RT for 30 min, the
reaction solution was poured into ice-chilled brine, washed with H2O and brine, dried
over MgSO4, filtered, and concentrated. Purification by flash chromatography (silica,
Et2O:hexanes = 1:2) provided compound 6 as an ivory solid (47%). 1H NMR (DMSO-d6,
300 MHz): δ 11.38 (1H, s), 10.87 (1H, s), 9.97 (1H, s), 7.59 (1H, s), 6.58 (1H, s). LRMS
(APCI+): Calc’d for C7H5ClO3 172.0 m/z, measured 173.1 (MH+).
To a suspension of compound 6 (0.65 g, 3.7 mmol), NaI (0.28 g, 1.9
mmol), and NaHCO3 (0.37g, 4.4 mmol) in MeCN (40 mL, 0.1 M) was
added benzyl bromide (0.45 mL, 3.7 mmol) at RT under argon. The
S10 reaction solution was stirred at 80 °C under argon for 16 h, cooled to RT, diluted with
EtOAc, washed with H2O and brine, dried over MgSO4, filtered, and concentrated.
Purification by flash chromatography (silica, EtOAc:hexanes = 1:10) provided compound
7 as a white solid (58%). 1H NMR (DMSO-d6, 300 MHz): δ 11.13 (1H, s), 10.02 (1H, s),
7.70 (1H, s), 7.49 – 7.34 (5H, m), 6.77 (1H, s), 5.27 (2H, s). LRMS (APCI+): Calc’d for
C14H11ClO3 262.0 m/z, measured 263.1 (MH+).
To a solution of compound 7 (2.4 g, 9.1 mmol), 4(dimethylamino)pyridine (DMAP, 0.29 g, 2.6 mmol), and
Boc-GABA-OH (2.2 g, 11 mmol) in dichloromethane (100
mL) was added N,N′-diisopropylcarbodiimide (DIC, 1.8 mL, 12 mmol) at RT under
argon. The reaction mixture was stirred at RT for 1.5 h, washed with H2O and brine,
dried over MgSO4, filtered, and concentrated. Purification by flash chromatography
(silica, EtOAc:hexanes = 1:3) provided compound 8 as a white solid (81%). 1H NMR
(DMSO-d6, 300 MHz): δ 9.90 (1H, s), 7.96 (1H, s), 7.50 – 7.37 (5H, m), 7.32 (1H, s),
6.95 (1H, t, J = 5.4 Hz), 5.30 (2H, s), 3.07 – 3.00 (2H, m), 2.68 (2H, t, J = 7.3 Hz), 1.81 –
1.72 (2H, m). LRMS (APCI+): Calc’d for C23H26ClNO6 447.1 m/z, measured 448.3 (MH+).
To a suspension of compound 8 (1.6 g, 3.6 mmol) and 3 Å
molecular sieves (powder, 3.0 g) in dry benzene (15 mL) was
added
2,8,9-triisopropyl-2,5,8,9-tetraaza-1-phosphabicyclo
[3.3.3]undecane (0.43 g, 1.4 mmol) in dry benzene (15 mL) via syringe at 40 °C under
argon. After being stirred at 50 °C for 17 h, the reaction mixture was diluted with CH2Cl2
(100 mL), filtered through celite, and concentrated to give a yellow solid. Purification by
flash chromatography (silica, EtOAc:hexanes = 1:4 → 1:3) provided compound 9 as an
ivory solid (44%). 1H NMR (CDCl3, 300 MHz): δ 7.46 – 7.28 (7H, m), 6.84 (1H, s), 5.17
(2H, s), 4.91 (1H, s), 3.42 – 3.36 (2H, m), 2.70 (2H, t, J = 5.7 Hz), 1.39 (9H, s). 13C NMR
(CDCl3, 75 MHz): δ 156.0, 153.1, 139.2, 135.3, 128.8, 128.4, 127.7, 127.2, 127.1, 124.4,
119.6, 113.4, 101.6, 79.3, 71.1, 39.1, 31.6, 28.4. LRMS (APCI+): Calc’d for
C23H24ClNO5 429.1 m/z, measured 430.8 (MH+).
S11 To compound 9 (0.28 g, 0.65 mmol) were added
trifluoroacetic acid (3.0 mL), H2O (0.15 mL), and
triisopropylsilane (0.15 mL). The reaction solution was
refluxed at 85 °C under argon for 16 h. After cooling to RT, Et2O (40 mL) was added to
the reaction solution to precipitate the crude product as a white solid. The emulsion was
centrifuged (4 °C, 3000 rpm, 5 min.), and ether was decanted. The crude solid was dried
under high vacuum before being dissolved in H2O/MeOH (4 mL/8 mL) and purified by
RP-HPLC using an appropriate linear gradient of methanol (A) and 0.1% (v/v) TFA/H2O
(B) (3–100% A over 25 min followed by an equilibrium back to 3% A). The fractions
containing the product (retention time ~16.2 min) were collected, concentrated, and
lyophilized to give Mini202 as a white solid (66% for two steps). 1H NMR (DMSO-d6,
300 MHz): δ 11.39 (1H, s), 7.76 – 7.72 (5H, m), 6.92 (1H, s), 3.06 (2H, t, J = 7.2 Hz),
2.72 (2H, t, J = 6.9 Hz).13C NMR (CD3OD, 75 MHz): δ 163.4, 157.8, 154.9, 142.8, 129.6,
122.0, 119.4, 114.0, 104.3, 39.4, 30.2. LRMS (APCI+): Calc’d for C11H10ClNO3 239.0 m/z,
measured 240.3 (MH+).
Scheme S4. Synthesis of Mini301, 302, and 401. (q) Methanesulfonic acid (MSA), RT, 1.5 h, 56%; (r)
MSA, RT, 2.5 h, 20%; (s) MSA, RT, 4 h, 5%.
S12 To a mixture of 2,4-dihroxybenzaldehyde 6 (50 mg, 0.36
mmol) and 1 (59 mg, 0.24 mmol) was added
methanesulfonic acid (1.0 mL) at 0 °C. The clear brown
solution gradually became dark orange within 1.5 h after which time the reaction mixture
was diluted with cold (−20 °C) ethyl ether (10 mL), and the crude mixture was extracted
into H2O (3 mL) and purified by RP-HPLC using an appropriate linear gradient of
acetonitrile containing 10% de-ionized water (A) and 0.1% (v/v) TFA/H2O (B) (3–50%
A over 20 min followed by a steep gradient to 100% A and equilibrium back to 3% A).
The fractions containing the product were collected, concentrated, and lyophilized to give
Mini301 as a white solid (56%). 1H NMR (DMSO-d6, 300 MHz): δ 11.37 (1H, s), 8.68
(1H, s), 7.85 (1H, d, J = 8.6 Hz), 7.75 (3H, bs), 6.89 (1H, dd, J = 8.6, 2.2 Hz), 6.79 (1H, d,
J = 2.1 Hz), 3.38 (2H, t, J = 6.8 Hz), 3.14 – 3.10 (2H, m).
13
C NMR (DMSO-d6, 75
MHz): δ 194.3, 164.8, 159.3, 157.5, 148.7, 133.1, 118.2, 114.6, 110.9, 102.0, 34.3.
LRMS (APCI+): Calc’d for C12H11NO4 233.1 m/z, measured 234.1 (MH+).
To a mixture of 5-chloro-2,4-dihroxybenzaldehyde (40
mg, 0.18 mmol) and 1 (29 mg, 0.12 mmol) was added
methanesulfonic acid (1.0 mL) at 0 °C. The clear brown
solution gradually became dark orange within 2.5 h after which time the reaction mixture
was diluted with cold (–20 °C) ethyl ether (10 mL), and the crude mixture was extracted
into H2O (3 mL) and purified by RP-HPLC using an appropriate linear gradient of
acetonitrile containing 10% de-ionized water (A) and 0.1% (v/v) TFA/H2O (B) (3–50%
A over 20 min followed by a steep gradient to 100% A and equilibrium back to 3% A).
The fractions containing the product were collected, concentrated, and lyophilized to give
Mini302 as a yellow solid (20%). 1H NMR (DMSO-d6, 400 MHz): δ 8.65 (1H, s), 8.11
(1H, s), 7.75 (3H, bs), 6.95 (1H, s), 3.38 (2H, t, J = 6.7 Hz), 3.14 – 3.07 (2H, m).
13
C
NMR (CD3OD, 75 MHz): δ 195.6, 161.3, 160.9, 157.5, 149.3, 132.6, 120.7, 120.5, 113.1,
104.0, 40.4, 35.9. LRMS (APCI+): Calc’d for C12H10ClNO4 267.0 m/z, measured 268.2
(MH+).
S13 To a mixture of 2,7-dihydroxynaphthalene (0.20 g, 1.3
mmol)
and
1
(0.20
mg,
0.83
mmol)
was
added
methanesulfonic acid (1.3 mL) at 0 °C. The clear brown
solution gradually became dark orange within 4 h after
which time the reaction mixture was diluted with cold (−20 °C) ethyl ether (10 mL),
centrifuged (3000 rpm) at 4 °C for 20 min. After removing the ether by aspiration, the
residual orange solid was dried under high vacuum, dissolved in H2O (3 mL), and
purified by RP-HPLC using an appropriate linear gradient of acetonitrile containing 10%
de-ionized water (A) and 0.1% (v/v) TFA/H2O (B) (3–50% A over 20 min followed by a
steep gradient to 100% A and equilibrium back to 3% A). The fractions containing the
product were collected, concentrated, and lyophilized to give Mini401 as a white solid
(5%). 1H NMR (DMSO-d6, 400 MHz): δ 10.21 (1H, s), 8.08 (1H, d, J = 8.9 Hz), 7.94
(1H, d, J = 8.8 Hz), 7.84 (3H, bs), 7.72 (1H, s), 7.33 (1H, d, J = 8.8 Hz), 7.18 (1H, dd, J =
8.8, 2.0 Hz), 6.44 (1H, s), 3.54 (2H, t, J = 6.8 Hz), 3.32 – 3.29 (2H, m).
13
C NMR
(DMSO-d6, 75 MHz): δ 159.2, 157.6, 155.1, 153.0, 133.9, 131.5, 130.8, 125.3, 117.0,
115.2, 114.0, 111.9, 107.9, 37.2, 33.9. LRMS (APCI+): Calc’d for C15H13NO3 255.1 m/z,
measured 256.2 (MH+).
Part II: HPLC Analysis of Coumarin Probes
As an estimate of the compounds’ purity, all the synthetic probes were analyzed by
analytical reverse phase HPLC (column is from GRACE VyDAC protein&peptide C18,
cat.# 218TP54) using an appropriate linear gradient of acetonitrile containing 10% deionized water (A) and 0.1% (v/v) TFA/H2O (B) (for Mini101–104, 107–109, and 201–
302: 3–30% A over 40 min followed by equilibrium back to 3% A, for Mini105, 106, and
401: 3–50% A over 40 min followed by equilibrium back to 3% A).
S14 Figure S2. Analytical RP-HPLC chromatograms of the coumarin probes. Detection at λabs = 254 nm.
S15 Part III: Photophysical Characterization and
Measurement of log D values
General
Ultraviolet absorption spectra were measured on a Molecular Devices SPECTRAmax
Plus 384 UV-Visible spectrophotometer operated through a Dell Pentium PC by
SOFTmax software. Fluorescence measurements (emission/excitation) were carried out
on a Jobin Yvon Fluorolog fluorescence spectrofluorometer.
Absorption
UV absorption spectra were taken by adding probe (2 µL of 10 mM stock solution in
DMSO) to 998 µL of PBS buffer at different pH values (final probe concn = 20 µM) in
quartz cuvette.
Emission/Excitation
Excitation/Emission spectra were taken by adding probe (20 µL of 0.1 mM stock solution
in distilled water) to 980 µL of PBS buffer of different pH values (final probe concn = 2
µM) in quartz cuvette.
pKa
The pKa values of probes were determined from the absorption spectra. The absorbance
ratio at two λabs, max was plotted versus pH of the PBS solution; the data were fit to a
sigmoid curve using KaleidaGraph (Synergy Software, Reading, PA) nonlinear
regression analysis program to determine the pKa value.
log D
S16 The log D values were determined by a traditional shake flask method. Each
measurement was performed in duplicate as follows. First, 20 µM probe solution in 1 mL
PBS (pH 7.4) was prepared to which 1 mL of n-octanol was added and mixed thoroughly.
The mixture was kept in dark for 3 days for complete equilibrium, and the concentrations
of probe in each layer were determined based on the UV absorbance. Log D values were
determined based on the following equation; log D = log[probe]oct – log[probe]PBS, where
[probe]oct and [probe]PBS are the concentrations of the probe in n-octanol and PBS,
respectively.
Table S1. Summary of photophysical properties, log D, and pKa values.
S17 Part IV: Protocols for Fluorescence Microscopy Assay in HEK and
VMAT2-HEK cells and Assay Results Summary
HEK GNTI- (nonglycosylating) cell line stably expressing VMAT2 (VMAT2-HEK) and
HEK GNTI- cell line stably transfected with TetR (HEK) to serve as a control were
kindly provided by the laboratory of Robert Edwards at UCSF. Cells were grown in
DMEM + Glutamax (Invitrogen #10569) with 10% fetal bovine serum (FBS) (Atlanta
Biologicals), 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen).
For fluorescence microscopy experiments, cells were plated on poly-D-lysine (Sigma
Aldrich, concn = 0.1 mg/mL) coated six-well dishes at a density of 1.0 x 105 cells per
well and grown at 37 °C in 5% CO2. After 5 days, the cells looked fibroblastic and had
reached ~70% confluence. The medium was removed by aspiration, and the cells were
washed with PBS (2 mL per well). To investigate the inhibitory effect of tetrabenazine
(TBZ) and reserpine, cells were incubated in 1 mL of experimental media (DMEM minus
phenol red (Invitrogen) with 4 mM L-glutamine (Invitrogen) and 1% charcoal/dextrantreated FBS (Atlanta Biologicals)) containing inhibitor (1 µM or 0 µM as a control,
prepared from 10 mM stock solution in DMSO) at 37 °C in 5% CO2 for 1 h. Then, the
probe uptake was initiated by adding 0.1 mL of experimental media containing probe
(220 µM, prepared from 10 mM stock solution in DMSO, final concn = 20 µM in the
uptake assay). After incubating at 37 °C for 30 min, the media was removed by aspiration,
and the cells were washed with PBS (2 mL per well) and treated with probe-free
experimental media. Fluorescence images were taken by using Leica FW 4000 equipped
with Chroma custom filter cube (ex = 350 ± 25 nm, em = 460 ± 25 nm) and Hamamatsu
digital camera C4742-95. The fluorescent images and bright field images were acquired
for 2000 ms and 37 ms, respectively. All images were adjusted using the same contrast
and brightness level using ImageJ (National Institute of Health).
For the chloroquine-induced Mini202 redistribution experiment, after probe loading and
cell washing with PBS, 1 mL of experimental media containing 300 µM chloroquine was
added to the cells at RT for 3 min, after which time fluorescent images were taken by the
same procedure described above.
S18 Figure S3. Summary of results from screening of Mini probes using VMAT2-HEK and HEK cells. Six
probes exhibited VMAT2-dependent uptake affording fluorescent puncta, i.e., they were taken up by
VMAT2-HEK cells but not control HEK cells and the uptake in VMAT2-HEK cells was abolished by
VMAT inhibitors tetrabenazine (TBZ) and reserpine (red signs). Three probes were taken up as fluorescent
puncta by both of VMAT2-HEK cells and HEK cells, regardless of the presence/absence of tetrabenazine or
reserpine (yellow signs). Five probes showed no uptake by either VMAT-HEK or HEK cells (black signs).
The observations summarized in Figure S3 indicated that probes Mini101–103, Mini106,
Mini201, and Mini202 are VMAT2 substrates. These probes are too polar to be
accumulated in HEK cells lacking VMAT2. In contrast, in cells where VMAT2 can
pump the probes into acidic organelles, we hypothesize that this transporter secondarily
facilitates the net influx across the plasma membrane of even hydrophilic probes via
passive diffusion. Compounds that are relatively lipophilic show non-selective uptake
(i.e., Mini104, 105, and 401). More polar compounds that are not VMAT2 substrates
show no uptake in either cell lines (Mini107–109, Mini301 and 302).
S19 Part V: Protocols for Fluorescence Microscopy in PC-12 cells
PC-12 cells were purchased and maintained according to the protocols provided by
American Type Culture Collection (ATCC, CRL-1721).5 PC-12 cells were grown in
RPMI-1640 (Invitrogen, #11875) with 10% horse serum (Invitrogen, #16050-114), 5%
fetal bovine serum (FBS) (Atlanta Biologicals), 100 U/ml penicillin/streptomycin
(Invitrogen). For fluorescence microscopy experiments, cells were plated on poly-Dlysine (Sigma Aldrich, concn = 0.1 mg/mL) coated six-well dishes at a density of 5.0 x
105 cells per well and grown at 37 °C in 5% CO2. After 6 days when the cells reached
~70% confluence, the medium was removed by aspiration, and the cells were washed
with PBS (2 mL per well). To investigate the inhibitory effect of reserpine, cells were
incubated in 1 mL of experimental media (RPMI-1640 minus phenol red supplemented
with 2 mM L-glutamine (Invitrogen #11835), 0.5% charcoal/dextran-treated FBS
(Atlanta Biologicals), 1 % charcoal/dextran-treated horse serum (Invitrogen), 100 U/ml
penicillin/streptomycin (Invitrogen)) containing reserpine (final concn = 1 µM prepared
from 10 mM stock solution in DMSO) at 37 °C in 5% CO2 for 1 h. Then, the probe
uptake was initiated by adding 0.1 mL of experimental media containing Mini202 (220
µM, prepared from 10 mM stock solution in DMSO, final concn = 20 µM in the uptake
assay) to the cells. After incubating the cells at 37 °C for 1 h, the medium was removed
by aspiration, and the cells were washed with PBS (2 mL per well) and treated with dyefree experimental media. Fluorescence images were acquired by using Leica FW 4000
equipped with Chroma custom filter cube (ex = 350 ± 25 nm, em = 460 ± 25 nm) and
Hamamatsu digital camera C4742-95. The fluorescent images and bright field images
were acquired for 2000 ms and 37 ms, respectively. All images were adjusted using the
same contrast and brightness level using ImageJ (National Institute of Health).
5 Among the two variations of PC-12 provided by ATCC, the experiments were carried out using the one
displaying loosely adherent (with no PDL coating) and multicellular-aggregating phenotype (CRL-1721).
S20 Part VI: Protocols for Two-photon Fluorescence Microscopy and pH
Measurement of Secretory Vesicles in PC-12 Cells
In Situ Calibration Curve
PC-12 cells were grown on 35×10 mm tissue culture dishes (Becton Dickinson Labware)
under the cell maintenance conditions as described in Part V. To calibrate Mini202
fluorescence intensity ratio by dual excitation (692 nm and 760 nm) for a range of pH
values in situ, vesicles of PC-12 cells were loaded with Mini202 by incubating the cells
with 20 µM Mini202 in 1 mL experiment medium for 1 h at 37 °C. After washing the
cells with dye-free PBS (1 mL per well), the extracellular media was replaced with 1mL
of calibration buffer of known pH in the presence of monensin, the Na+/H+ ionophore,
and nigericin, the K+/H+ ionophore, which act to equilibrate the pH of cytosol and
vesicles with that of extracellular media. This pH calibration buffer contains 125 mM
KCl, 20 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 5 µM nigericin, 5 µM monensin, and
25 mM buffer (acetate for pH 4.27, 4.69, 5.39; MES for pH 5.74, 6.15; HEPES for pH
6.41, 6.95, 7.52). The cells were treated with the calibration buffer for 8–10 min at RT,
and fluorescent images were acquired by a two-photon fluorescence microscope (Prairie
Ultima multiphoton microscope operated with Prairie View 3.0.0.3 software for scan
control and image collection (Prairie Technologies, Middleton, WI) with Mai Tai HP
Ti:sapphire laser (Spectra-Physics, Newport Instruments, Irvine, CA) (excitation 692 or
760 nm, emission 430–510 nm) and water-immersion, IR-corrected objective from
Olympus designated LUMPlanFl/IR 60x/0.90 NA on an Olympus BX61W1 microscope).
For each 35 mm plate, two pairs of fluorescent images by 692 nm and 760 nm excitation
were collected at a given pH value, and this process was repeated at least 2–3 times using
a new plate of cells each time. The ratio of fluorescence intensity by excitation at 760 nm
and 692 nm (I760/I692) was plotted versus pH of the calibration solution; the data were fit
to a sigmoid curve using KaleidaGraph (Synergy Software, Reading, PA) nonlinear
regression analysis program to construct a calibration curve (Figure 6A in the manuscript,
ratio(760/692) = 0.09+1.43/(1+(pH/a)^b); a = 5.91; b = −16.10; R2 = 0.98). The
calibraion curve was constructed three times on three different days to determine the in
S21 situ pKa value of Mini202 in the LDCVs of PC-12 cells (pKa = 5.93 ± 0.04, n = 3).
Ratiometric pH measurements of LDCVs in PC-12 cells in situ
After PC-12 cells were incubated with 20 µM Mini202 for 1 h at 37 °C in 5% CO2,
fluorescent images containing ~10 cells were acquired by dual excitation (692 nm and
760 nm) using the Prairie multiphoton microscope, as described above. Fluorescence
signals from LDCVs were manually selected using Volocity version 4.4 software (Perkin
Elmer, Waltham, MA) with an appropriate object-selection parameter settings (e.g., the
object size and fluorescence intensity). At this step, the total area and the punctate pattern
selected as LDCVs from the two images (by 692 nm and 760 nm) were assured to be
similar each other. Mean fluorescence intensity of the LDCVs from 7–10 cells were
determined, which in turn was used to get fluorescence intensity ratio I760/I692. This
process was repeated three times in duplicate, using new plate of cells for each
measurement.
In order to measure the pH change induced by methamphetamine, the PC-12 cells
preloaded with Mini202 were washed with PBS, treated with 1mL of experimental media
containing 100 µM methamphetamine (prepared from 50 mM stock solution in DMSO)
for 5 min at RT, and imaged by the Prairie multiphoton microscope. Methamphetamine
a
b
c
Figure S4. Two-photon fluorescence image of PC-12 cells treated with 100 μM methamphetamine for 5
min at RT with two-photon excitation (a) at 692 nm and (b) at 760 nm. (c) Pseudocolor image of I760/I692
and corresponding pH values. The vesicular pH in PC-12 cells increased from 5.9 to 6.4 by the effect of
methamphetamine.
S22 treatment resulted in diffusion of Mini202 from the vesicles to cytoplasm to some extent,
however, only fluorescence signals from LDCVs were selected as objects and taken
account into the pH calculation (Figure S4).
Part VII: Preparation and Uptake Test of the Hydrochloride Salt of
Mini202
In order to avoid possible toxic effects of trifluoroacetic acid present with Mini202 as a
TFA salt, we prepared Mini202 as a hydrochloride salt from the corresponding TFA salt
as described below. Mini202•TFA (20 mg) was dissolved in mixture of 0.8 M HCl
aqueous solution (5 mL) and methanol (0.2 mL). The cloudy solution became clear after
being heated at 80 °C for 5 min. The solution was cooled down to RT and lyophilized to
afford Mini202•HCl as a white solid (12 mg). The purity of the compound was checked
by 1H nmr spectroscopy, analytical reverse-phase HPLC, and mass spectrometry. 19F nmr
was obtained to confirm the absence of TFA in the Mini202•HCl salt.
The activity of Mini202•HCl salt toward VMAT2 was tested in VMAT2-HEK and HEK
cells by the same method described in Part IV. Both Mini202•TFA and Mini202•HCl
salts showed the same pattern of punctate uptake in a VMAT2-dependent manner. This
uptake was abolished in the presence of VMAT2 inhibitors (1 µM TBZ or 1 µM
reserpine), which suggests the salt conversion from TFA to HCl does not affect
Mini202’s activity as a VMAT2 substrate in VMAT2-HEK cells (Figure S5).
S23 a b c d e f g h i j k l Figure S5. Uptake study of Mini202•TFA (a–f) and Mini202•HCl (g–l) in VMAT2-HEK cells and HEK
cells. 20 µM Mini202•TFA or Mini202•HCl was incubated in VMAT2-HEK cells for 30 min to afford
fluorescent puncta (a, g), and preincubation of the VMAT2-HEK cells with VMAT2 inhibitor TBZ (b, h) or
reserpine (c, i) abolished the uptake of Mini202•TFA and Mini202•HCl. In HEK cells, both salt forms of
Mini202 showed no uptake in the absence (d, j) or presence of TBZ (e, k) and reserpine (f, l). λex = 350 ±
25 nm, λem = 460 ± 25 nm.
S24 Part VIII: 1H and 13C NMR of Coumarin Probes
S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38